Project Summary/Abstract The overwhelming public health burden of HPV-associated head and neck squamous cell carcinoma has created great demand for novel, broadly effective therapies with reduced treatment morbidity and improved long-term survival. The generation of tumor-specific CD8+ T cell immunity requires potent antigen cross-presentation by dendritic cells (DCs) since tumor cells do not efficiently present relevant CD8+ T cell epitopes. cDC1s are known for their ability to process exogenous antigen and potently cross-present to CD8+ T cells. Because of this, innovative strategies to enhance cDC1s could robustly induce HPV-specific immunity and have great therapeutic potential in the treatment of HPV-associated HNC. FMS-like tyrosine kinase 3 ligand (Flt3L) is a cytokine that expands and differentiates DC precursors to murine cDC1s, but therapeutic potential of Flt3L is limited because of its short half-life and global distribution in vivo. We have overcome the described issues of Flt3L by generating a genetic fusion of Albumin (Alb) to Flt3L, named Albumin-Flt3L (Alb-Flt3L). Alb has a long half-life due to FcRn- mediated transcytolic recycling and exhibits selective trafficking to the LN and TM. Once cDC1s expand, they require a strong, tissue localized source of inflammation for activation, or they will present T cell epitopes without adequate costimulation, causing suppression. Enteric bacteria such as Salmonella serve as an ideal agent for tumor specific cDC1 activation as they can provide numerous pathogen-associated molecular patterns for cDC1 activation, and have been described to colonize the tumor efficiently likely due to hypoxia. One caveat of bacteria therapy in cancer is that consideration must be made regarding preclinical model selection, as colonization varies greatly between spontaneous and transplantable tumor models. Our preliminary data shows that Alb-Flt3L fusion protein exhibits increased half-life and selective accumulation in LN and tumor compared to native Flt3L. Alb- Flt3L is able to expand cross-presenting DC populations in vivo following a single injection and leads to enhanced HPV-specific T cells in a vaccination model and tumor control in a proof-of-principle experiment. The Salmonella strain used in our experiments successfully colonizes the tumor and can efficiently activate Alb-Flt3L-derived DCs. In this proposal, the ability of Alb-Flt3L + Salmonella to promote HPV-specific cytotoxic T cell antitumor immunity through the expansion and activation of cross-presenting DCs and subsequent tumor control in our spontaneous and transplantable HPV-associated HNC models will be investigated. HPV-specific CD8+ T cell responses will be evaluated using tetramer and TCR sequencing approaches. The mechanism by which Alb- Flt3L mediates its immunostimulatory function will also be interrogated using appropriate deficient mouse models. Studies regarding colonization of Salmonella in spontaneous vs transplantable tumor models will be investigated using our high-resolution nanoPET/CT system and appropriate PET tracers. Successful completion of this proposal will generate valuable preclinical and mechanistic data regarding the therapeutic potential of Alb- Flt3L + Salmonella as a universal strategy to treat HPV-associated HNC as well as many other types of cancer.